Novel Strategies and Tools for Enhanced Sensitivity in Routine Biomolecule Analytics

Abstract

The combination of flexible and robust immunoassay technology with the sensitivity of DNA as a signal amplification template is realized in Immuno-PCR ("IPCR"). Classical ELISA is converted to IPCR by exchange of antibodyenzyme detection conjugates with antibody-DNA conjugates. The value of ultra-sensitive analytics deriving from this for pharmaceutical R&D is threefold: (I) Deeper understanding of biomolecular interactions for the development of new compounds. (II) Monitoring of compounds at very low concentrations in toxicokinetic and pharmacokinetic clinical studies. (III.) Control of compound functionality and therapeutic effects by surveillance of characteristic biomarkers and/or immunogenicity reactions. This review summarizes background information about general selection of IPCR targets, actual standard assay procedures and work with biological matrices. Quantitative real-time detection as well as optimized reagents and protocols revealed a typically 100-10,000-fold increase in sensitivity and a broad dynamic range compared to ELISA. Case studies are discussed for (I) the analysis of biomolecular interaction with proximity ligation technologies and IPCR, (II) pharmacokinetic studies of novel drugs with validation data for assay precision and recovery and (III) biomarker profiling, including cytokine multiplex assays and polyplex trace analysis in extremely small sample volumes. In a survey of results from recent innovations, the potential of this emerging field of applications is evaluated for novel pathways in study design and analytics.

Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques

“…4a). While the relatively young technology of DNA-enhanced LBAs has previously been surveyed, [44][45][46][47][48][49][50][51][52] we will here discuss this technology in the context of other emerging techniques for high-sensitivity analyses of biomacromolecules with an emphasis on case studies from preclinical and clinical sample testing in support of pharmaceutical drug development programs. In contrast, the so-called DNA-enhanced LBAs usually employ polymerase-chain reaction (PCR) 43 for signal amplification, thus enabling the exponential amplification of marker molecules.…”

“…This background signal needs to be compensated by an increase in specific signals, which can be obtained by tailored SGR with increased affinity, such as antibody-DNA conjugates. 61 Negative influences of the biological matrix can be minimized by dilution of samples under optimized buffer conditions 45,49,66,[77][78][79] because dilution affects low affinity interactions, i.e., the non-specific binding of assays reagents to matrix components, to a greater extent than the high-affinity interactions between the SGR and the target. [74][75][76] Importantly, the composition and complexity of the biological matrix often varies to a large extent from, e.g., species to species, between different study cohorts, healthy to diseased individuals, or even between samples drawn at different time points from one individual.…”

“…Because these developments were typically conducted as proof-of-concept studies and they have been surveyed previously, 45,47,51,52,129 we will here focus on the most important features of IPCR and illustrate the state-of-the-art of this LBA technology on the basis of case studies in the area of pharmaceutical drug development programs. 49 Various modifications of the initial IPCR protocol 128 have been reported, many of which have led to a significant increase in the analytical sensitivity. Due to the steady-state on/off kinetic nature of antibody-antigen binding, a level of approx.…”

Emerging Technologies to Increase Ligand Binding Assay Sensitivity

“…IPCR case studies demonstrate sensitivities close to the theoretical detection limit of LBAs, down to a few thousand analyte molecules and wide dynamic range of up to five logs (9,(28)(29)(30)(31). Given a protein of 50-100 kD in size, this corresponds to LLOQ levels in the sub-picogram per milliliter levels in biological samples (8,32). Typical sample volumes are 15-30 μL for duplicates.…”

“…The concepts of IPCR were initially described in 1992 (7). However, the commercially available Imperacer® IPCR platform from Chimera Biotec was introduced recently and provides optimized reagents and pre-synthesized DNA-detector conjugates (8,9) for best assay performance in a 96-well plate format.…”